6-amino-2--9-[5-(1-piperidinyl)-7,9-dihydro-8h-purin-8-one maleate

ABSTRACT

The present invention relates to a compound which is 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one: 
     
       
         
         
             
             
         
       
     
     in the form of a maleate salt, may be useful in the treatment of various disorders, for example the treatment of allergic diseases and other inflammatory conditions for example allergic rhinitis and asthma, the treatment of infectious diseases and cancer, and may also be useful as vaccine adjuvants.

BACKGROUND OF THE INVENTION

The present invention relates to compounds, processes for theirpreparation, compositions containing them, to their use in the treatmentof various disorders in particular allergic diseases and otherinflammatory conditions for example allergic rhinitis and asthma,infectious diseases, cancer, and as vaccine adjuvants.

Vertebrates are constantly threatened by the invasion of microorganismsand have evolved mechanisms of immune defence to eliminate infectivepathogens. In mammals, this immune system comprises two branches; innateimmunity and acquired immunity. The first line of host defence is theinnate immune system, which is mediated by macrophages and dendriticcells. Acquired immunity involves the elimination of pathogens at thelate stages of infection and also enables the generation ofimmunological memory. Acquired immunity is highly specific, due to thevast repertoire of lymphocytes with antigen-specific receptors that haveundergone gene rearrangement.

The innate immune response was originally thought to be non-specific,but is now known to be able to discriminate between self and a varietyof pathogens. The innate immune system recognises microbes via a limitednumber of germline-encoded Pattern-Recognition Receptors (PRRs) whichhave a number of important characteristics.

Toll-like receptors (TLRs) are a family of ten Pattern RecognitionReceptors described in man. TLRs are expressed predominantly by innateimmune cells where their role is to monitor the environment for signs ofinfection and, on activation, mobilise defence mechanisms aimed at theelimination of invading pathogens. The early innate immune-responsestriggered by TLRs limit the spread of infection, while thepro-inflammatory cytokines and chemokines that they induce lead torecruitment and activation of antigen presenting cells, B cells, and Tcells. The TLRs can modulate the nature of the adaptive immune-responsesto give appropriate protection via dendritic cell-activation andcytokine release (Akira S., et al, Nat. Immunol., 2001: 2, 675-680). Theprofile of the response seen from different TLR agonists depends on thecell type activated.

TLR7 is a member of the subgroup of TLRs (TLRs 3, 7, 8, and 9),localised in the endosomal compartment of cells which have becomespecialised to detect non-self nucleic acids. TLR7 plays a key role inanti-viral defence via the recognition of ssRNA (Diebold S. S., et al,Science, 2004: 303, 1529-1531; and Lund J. M., et al, PNAS, 2004: 101,5598-5603). TLR7 has a restricted expression-profile in man and isexpressed predominantly by B cells and plasmacytoid dendritic cells(pDC), and to a lesser extent by monocytes. Plasmacytoid DCs are aunique population of lymphoid-derived dendritic cells (0.2-0.8% ofPeripheral Blood Mononuclear Cells (PBMCs)) which are the primary type Iinterferon-producing cells secreting high levels of interferon-alpha(IFNα) and interferon-beta (IFNβ) in response to viral infections (LiuY-J, Annu. Rev. Immunol., 2005: 23, 275-306).

Allergic diseases are associated with a Th2-biased immune-response toallergens. Th2 responses are associated with raised levels of IgE,which, via its effects on mast cells, promotes a hypersensitivity toallergens, resulting in the symptoms seen, for example, in allergicrhinitis. In healthy individuals the immune-response to allergens ismore balanced with a mixed Th2/Th1 and regulatory T cell response. TLR7ligands have been shown to reduce Th2 cytokine and enhance Th1 cytokinerelease in vitro and to ameliorate Th2-type inflammatory responses inallergic lung models in vivo (Fili L., et al, J. All. Clin. Immunol.,2006: 118, 511-517; Moisan J., et al, Am. J. Physiol. Lung Cell Mol.Physiol., 2006: 290, L987-995; Tao et al, Chin. Med. J., 2006: 119,640-648). Thus, TLR7 ligands have the potential to rebalance theimmune-response seen in allergic individuals and lead to diseasemodification.

Central to the generation of an effective innate immune response inmammals are mechanisms which bring about the induction of interferonsand other cytokines which act upon cells to induce a number of effects.These effects can include the activation of anti-infective geneexpression, the activation of antigen presentation in cells to drivestrong antigen-specific immunity and the promotion of phagocytosis inphagocytic cells.

Interferon was first described as a substance which could protect cellsfrom viral infection (Isaacs & Lindemann, J. Virus Interference. Proc.R. Soc. Lon. Ser. B. Biol. Sci. 1957: 147, 258-267). In man, the type Iinterferons are a family of related proteins encoded by genes onchromosome 9 and encoding at least 13 isoforms of interferon alpha(IFNα) and one isoform of interferon beta (IFNβ). Recombinant IFNα wasthe first approved biological therapeutic and has become an importanttherapy in viral infections and in cancer. As well as direct antiviralactivity on cells, interferons are known to be potent modulators of theimmune response, acting on cells of the immune system.

As a first-line therapy for hepatitis C virus (HCV) disease, interferoncombinations can be highly effective at reducing viral load and in somesubjects in eliminating viral replication. However, many patients failto show a sustained viral response and in these patients viral load isnot controlled. Additionally, therapy with injected interferon may beassociated with a number of unwanted adverse effects which are shown toaffect compliance (Dudley T., et al, Gut., 2006: 55(9), 1362-3).

Administration of a small molecule compound which could stimulate theinnate immune response, including the activation of type I interferonsand other cytokines, could become an important strategy for thetreatment or prevention of human diseases including viral infections.This type of immunomodulatory strategy has the potential to identifycompounds which may be useful not only in infectious diseases but alsoin cancer (Krieg., Curr. Oncol. Rep., 2004: 6(2), 88-95), allergicdiseases (Moisan J., et al, Am. J. Physiol. Lung Cell Mol. Physiol.,2006: 290, L987-995), other inflammatory conditions such as irritablebowel disease (Rakoff-Nahoum S., Cell., 2004, 23, 118(2): 229-41), andas vaccine adjuvants (Persing et al., Trends Microbiol., 2002: 10(10Suppl), S32-7).

In animal models, imiquimod demonstrated adjuvant activities eithertopically (Adams S., et al, J. Immunol., 2008, 181:776-84; Johnston D.,et al, Vaccine, 2006, 24:1958-65), or systemically (Fransen F. et al,Infect. Immun., 2007, 75:5939-46). Resiquimod and other related TLR7/8agonists have also been shown to display adjuvant activity (Ma R. et al,Biochem. Biophys. Res. Commun., 2007, 361:537-42; Wille-Reece U., et al,Proc. Natl. Acad. Sci. USA, 2005, 102:15190-4; Wille-Reece U., et al,US2006045885 A1).

Mechanisms which lead to induction of type I interferons are only partlyunderstood. One mechanism which can lead to the induction of interferonin many cell types is the recognition of double-stranded viral RNA bythe RNA helicases RIG-I and MDA5. This mechanism is thought to be theprimary mechanism by which interferons are induced by Sendai virusinfection of cells.

Further mechanisms for the induction of interferons are viaTLR-dependent signalling events. In man, plasmacytoid dendritic cells(pDCs) are professional interferon-producing cells, able to make largeamounts of interferons in response to, for example, viral infection.These pDCs are shown to preferentially express TLR7 and TLR9 andstimulation of these receptors with viral RNA or DNA respectively caninduce expression of interferon alpha.

Oligonucleotide agonists of TLR7 and TLR9, and small moleculepurine-based agonists of TLR7 have been described which can induceinterferon alpha from these cell types in animals and in man (Takeda K.et al, Annu. Rev. Immunol., 2003: 21, 335-76). TLR7 agonists includeimidazoquinoline compounds such as imiquimod and resiquimod, oxoadenineanalogues and also nucleoside analogues such as loxoribine and7-thia-8-oxoguanosine which have long been known to induce interferonalpha. International Patent Application publication number WO2008/114008 (AstraZeneca AB/Dainippon Sumitomo Pharma Co. Ltd.)discloses 9-substituted-8-oxoadenine compounds as TLR7 modulators.

It remains unclear how small molecule purine-like compounds can inducetype I interferons and other cytokines since the molecular targets ofthese known inducers have not been identified. However, an assaystrategy has been developed to characterise small molecule inducers ofhuman interferon IFNα (regardless of mechanism) which is based onstimulation of primary human donor cells with compounds, and isdisclosed herein.

BRIEF DESCRIPTION OF THE INVENTION

The compound6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,which is disclosed in International Application No. PCT/EP2009/060265,published as WO2010/018133, has been shown to be an inducer of humaninterferon and may possess an improved profile with respect to knowninducers of human interferon, for example enhanced potency, and may showenhanced selectivity for IFNα with respect to TNFα. It is expected that6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt will be more easily formulated and/orprocessed and/or handled. For example, the purity of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onemay be improved via formation and/or recrystallisation of a maleatesalt, and/or its stability of may be improved vis-à-vis the free base.6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onewhich induces human interferon may be useful in the treatment of variousdisorders, for example the treatment of allergic diseases and otherinflammatory conditions for example allergic rhinitis and asthma, thetreatment of infectious diseases and cancer, and may also be useful as avaccine adjuvant. It is expected that6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt will have similar pharmacologicalproperties.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneis a potent immunomodulator and accordingly, care should be exercised inits handling.

SUMMARY OF THE INVENTION

In a first aspect, there is provided a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one:

in the form of a maleate salt.

Further, there is provided6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt, in which the ratio of maleate anion to6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneis 1:1.

There is thus provided, as a further aspect of the invention, a compoundwhich is 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, for use in therapy.

There is also therefore provided a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, for use in the treatment of allergicdiseases and other inflammatory conditions, infectious diseases, andcancer.

There is also therefore provided a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, for use in the treatment of allergicrhinitis.

There is also therefore provided a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, for use in the treatment of asthma.

There is also therefore provided a vaccine adjuvant comprising acompound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt.

There is further provided an immugenic composition comprising an antigenor antigen composition and a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt.

There is further provided a vaccine composition comprising an antigen orantigen composition and a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt.

There is further provided a method of treating or preventing diseasecomprising the administration to a human subject suffering from orsusceptible to disease, an immugenic composition comprising an antigenor antigen composition and a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt.

There is further provided a method of treating or preventing diseasecomprising the administration to a human subject suffering from orsusceptible to disease, a vaccine composition comprising an antigen orantigen composition and a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt.

There is further provided the use of a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, for the manufacture of an immugeniccomposition comprising an antigen or antigen composition, for thetreatment or prevention of disease.

There is further provided the use of a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, for the manufacture of a vaccinecomposition comprising an antigen or antigen composition, for thetreatment or prevention of disease.

There is further provided the use of a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, for the manufacture of a medicament forthe treatment of allergic diseases and other inflammatory conditions,infectious diseases, and cancer.

There is further provided the use of a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, for the manufacture of a medicament forthe treatment of allergic rhinitis.

There is further provided the use of a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, for the manufacture of a medicament forthe treatment of asthma.

There is further provided a method of treatment of allergic diseases andother inflammatory conditions, infectious diseases, and cancer, whichmethod comprises administering to a human subject in need thereof, atherapeutically effective amount of a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt.

There is further provided a method of treatment of allergic rhinitis,which method comprises administering to a human subject in need thereof,a therapeutically effective amount of a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt.

There is further provided a method of treatment of asthma, which methodcomprises administering to a human subject in need thereof, atherapeutically effective amount of a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt.

The invention provides in a further aspect, a combination comprising acompound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, together with at least one othertherapeutically-active agent.

There is further provided a pharmaceutical composition comprising acompound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, and one or more pharmaceuticallyacceptable diluents or carriers.

There is also provided a process for preparing a pharmaceuticalcomposition which comprises admixing a compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, with one or more pharmaceuticallyacceptable diluents or carriers.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,and salts thereof may be prepared by the methodology described in U.S.Provisional Application No. 61/087,777 and International Application No.PCT/EP2009/060265, published as WO2010/018133, incorporated herein byreference

DETAILED DESCRIPTION OF THE INVENTION

It will be appreciated that many organic compounds can form complexeswith solvents in which they are reacted or from which they areprecipitated or crystallised. These complexes are known as “solvates”.For example, a complex with water is known as a “hydrate”. Solvents withhigh boiling points and/or solvents with a high propensity to formhydrogen bonds such as water, ethanol, iso-propyl alcohol, and N-methylpyrrolidinone may be used to form solvates. Methods for theidentification of solvates include, but are not limited to, NMR andmicroanalysis. Solvates of the6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt are within the scope of the invention.

It will be appreciated that6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onewill be present primarily in the form of the S-isomer, but may includesmall amounts, for example less than 5%, or less than 3%, and preferablyless than 1%, or preferably less than 0.5% of the R-isomer. It will beappreciated that maleate salts of these mixtures are considered withinthe scope of the present invention.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may exist in tautomeric forms. It will beunderstood that the present invention encompasses all of the tautomersof6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt, whether as individual tautomers or asmixtures thereof.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may be in crystalline or amorphous form.Furthermore, some of the crystalline forms of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may exist as polymorphs, which areincluded within the scope of the present invention. The mostthermodynamically stable polymorphic form or forms are of particularinterest.

Polymorphic forms of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may be characterised and differentiatedusing a number of conventional analytical techniques, including, but notlimited to, X-ray powder diffraction (XRPD), infrared spectroscopy (IR),Raman spectroscopy, differential scanning calorimetry (DSC),thermogravimetric analysis (TGA) and solid-state nuclear magneticresonance (ssNMR).

It will be appreciated from the foregoing that included within the scopeof the invention are solvates, hydrates, isomers and polymorphic formsof6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt.

Examples of disease states in which6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may have potentially beneficial effectsinclude allergic diseases and other inflammatory conditions for exampleallergic rhinitis and asthma, infectious diseases, and cancer.6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt is also of potential use as a vaccineadjuvant.

As a modulator of the immune response,6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may also be useful, as stand-alone or incombination as an adjuvant, in the treatment and/or prevention ofimmune-mediated disorders, including but not limited to inflammatory orallergic diseases such as asthma, allergic rhinitis andrhinoconjuctivitis, food allergy, hypersensitivity lung diseases,eosinophilic pneumonitis, delayed-type hypersensitivity disorders,atherosclerosis, pancreatitis, gastritis, colitis, osteoarthritis,psoriasis, sarcoidosis, pulmonary fibrosis, respiratory distresssyndrome, bronchiolitis, chronic obstructive pulmonary disease,sinusitis, cystic fibrosis, actinic keratosis, skin dysplasia, chronicurticaria, eczema and all types of dermatitis.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt, may also be useful in the treatmentand/or prevention of reactions against respiratory infections, includingbut not limited to airways viral exacerbations and tonsillitis.6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may also be useful in the treatment and/orprevention of autoimmune diseases including but not limited torheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus,Sjöegrens disease, ankylosing spondylitis, scleroderma, dermatomyositis,diabetes, graft rejection, including graft-versus-host disease,inflammatory bowel diseases including, but not limited to, Crohn'sdisease and ulcerative colitis.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may also be useful in the treatment ofinfectious diseases including, but not limited to, those caused byhepatitis viruses (e.g. hepatitis B virus, hepatitis C virus), humanimmunodeficiency virus, papillomaviruses, herpesviruses, respiratoryviruses (e.g. influenza viruses, respiratory syncytial virus,rhinovirus, metapneumovirus, parainfluenzavirus, SARS), and West Nilevirus.6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may also be useful in the treatment ofmicrobial infections caused by, for example, bacteria, fungi, orprotozoa. These include, but are not limited to, tuberculosis, bacterialpneumonia, aspergillosis, histoplasmosis, candidosis, pneumocystosis,leprosy, chlamydia, cryptococcal disease, cryptosporidosis,toxoplasmosis, leishmania, malaria, and trypanosomiasis.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may also be useful in the treatment ofvarious cancers, in particular the treatment of cancers that are knownto be responsive to immunotherapy and including, but not limited to,renal cell carcinoma, lung cancer, breast cancer, colorectal cancer,bladder cancer, melanoma, leukaemia, lymphomas and ovarian cancer.

It will be appreciated by those skilled in the art that referencesherein to treatment or therapy may, depending on the condition, extendto prophylaxis as well as the treatment of established conditions.

As mentioned herein,6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one in the form of a maleate salt may beuseful as therapeutic agent.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may be formulated for administration inany convenient way.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may, for example, be formulated for oral,topical, inhaled, intranasal, buccal, parenteral (for exampleintravenous, subcutaneous, intradermal, or intramuscular) or rectaladministration. In one aspect,6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt is formulated for oral administration. Ina further aspect,6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt is formulated for topical administration,for example intranasal or inhaled administration.

Tablets and capsules for oral administration may contain conventionalexcipients such as binding agents, for example syrup, acacia, gelatin,sorbitol, tragacanth, mucilage of starch, cellulose or polyvinylpyrrolidone; fillers, for example, lactose, microcrystalline cellulose,sugar, maize starch, calcium phosphate or sorbitol; lubricants, forexample, magnesium stearate, stearic acid, talc, polyethylene glycol orsilica; disintegrants, for example, potato starch, croscarmellose sodiumor sodium starch glycollate; or wetting agents such as sodium laurylsulfate. The tablets may be coated according to methods well known inthe art.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may contain conventionaladditives such as suspending agents, for example, sorbitol syrup, methylcellulose, glucose/sugar syrup, gelatin, hydroxymethyl cellulose,carboxymethyl cellulose, aluminium stearate gel or hydrogenated ediblefats; emulsifying agents, for example, lecithin, sorbitan mono-oleate oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters, propyleneglycol or ethyl alcohol; or preservatives, for example, methyl or propylp-hydroxybenzoates or sorbic acid. The preparations may also containbuffer salts, flavouring, colouring and/or sweetening agents (e.g.mannitol) as appropriate.

Compositions for intranasal administration include aqueous compositionsadministered to the nose by drops or by pressurised pump. Suitablecompositions contain water as the diluent or carrier for this purpose.Compositions for administration to the lung or nose may contain one ormore excipients, for example one or more suspending agents, one or morepreservatives, one or more surfactants, one or more tonicity adjustingagents, one or more co-solvents, and may include components to controlthe pH of the composition, for example a buffer system. Further, thecompositions may contain other excipients such as antioxidants, forexample sodium metabisulfite, and taste-masking agents. Compositions mayalso be administered to the nose or other regions of the respiratorytract by nebulisation.6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may offer sufficient solubility andstability for presentation as an aqueous intranasal solutionformulation.

Intranasal compositions may permit6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt to be delivered to all areas of the nasalcavities (the target tissue) and further, may permit the activecompounds to remain in contact with the target tissue for longer periodsof time. A suitable dosing regime for intranasal compositions would befor the patient to inhale slowly through the nose subsequent to thenasal cavity being cleared. During inhalation the composition would beadministered to one nostril while the other is manually compressed. Thisprocedure would then be repeated for the other nostril. Typically, oneor two sprays per nostril would be administered by the above procedureone, two, or three times each day, ideally once daily. Of particularinterest are intranasal compositions suitable for once-dailyadministration.

The suspending agent(s), if included, will typically be present in anamount of from 0.1 to 5% (w/w), such as from 1.5% to 2.4% (w/w), basedon the total weight of the composition. Examples of pharmaceuticallyacceptable suspending agents include, but are not limited to, Avicel®(microcrystalline cellulose and carboxymethylcellulose sodium),carboxymethylcellulose sodium, veegum, tragacanth, bentonite,methylcellulose, xanthan gum, carbopol and polyethylene glycols.

Compositions for administration to the lung or nose may contain one ormore excipients may be protected from microbial or fungal contaminationand growth by inclusion of one or more preservatives. Examples ofpharmaceutically acceptable anti-microbial agents or preservativesinclude, but are not limited to, quaternary ammonium compounds (forexample benzalkonium chloride, benzethonium chloride, cetrimide,cetylpyridinium chloride, lauralkonium chloride and myristyl picoliniumchloride), mercurial agents (for example phenylmercuric nitrate,phenylmercuric acetate and thimerosal), alcoholic agents (for examplechlorobutanol, phenylethyl alcohol and benzyl alcohol), antibacterialesters (for example esters of para-hydroxybenzoic acid), chelatingagents such as disodium edetate (EDTA) and other anti-microbial agentssuch as chlorhexidine, chlorocresol, sorbic acid and its salts (such aspotassium sorbate) and polymyxin. Examples of pharmaceuticallyacceptable anti-fungal agents or preservatives include, but are notlimited to, sodium benzoate, sorbic acid, sodium propionate,methylparaben, ethylparaben, propylparaben and butylparaben. Thepreservative(s), if included, may be present in an amount of from 0.001to 1% (w/w), such as from 0.015% to 0.5% (w/w) based on the total weightof the composition.

Compositions (for example wherein at least one compound is insuspension) may include one or more surfactants which functions tofacilitate dissolution of the medicament particles in the aqueous phaseof the composition. For example, the amount of surfactant used is anamount which will not cause foaming during mixing. Examples ofpharmaceutically acceptable surfactants include fatty alcohols, estersand ethers, such as polyoxyethylene (20) sorbitan monooleate(Polysorbate 80), macrogol ethers, and poloxamers. The surfactant may bepresent in an amount of between about 0.01 to 10% (w/w), such as from0.01 to 0.75% (w/w), for example about 0.5% (w/w), based on the totalweight of the composition.

One or more tonicity-adjusting agent(s) may be included to achievetonicity with body fluids e.g. fluids of the nasal cavity, resulting inreduced levels of irritancy. Examples of pharmaceutically acceptabletonicity-adjusting agents include, but are not limited to, sodiumchloride, dextrose, xylitol, calcium chloride, glucose, glycerine andsorbitol. A tonicity-adjusting agent, if present, may be included in anamount of from 0.1 to 10% (w/w), such as from 4.5 to 5.5% (w/w), forexample about 5.0% (w/w), based on the total weight of the composition.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may be buffered by the addition ofsuitable buffering agents such as sodium citrate, citric acid,trometamol, phosphates such as disodium phosphate (for example thedodecahydrate, heptahydrate, dihydrate and anhydrous forms), or sodiumphosphate and mixtures thereof.

A buffering agent, if present, may be included in an amount of from 0.1to 5% (w/w), for example 1 to 3% (w/w) based on the total weight of thecomposition.

Examples of taste-masking agents include sucralose, sucrose, saccharinor a salt thereof, fructose, dextrose, glycerol, corn syrup, aspartame,acesulfame-K, xylitol, sorbitol, erythritol, ammonium glycyrrhizinate,thaumatin, neotame, mannitol, menthol, eucalyptus oil, camphor, anatural flavouring agent, an artificial flavouring agent, andcombinations thereof.

One or more co-solvent(s) may be included to aid solubility of themedicament compound(s) and/or other excipients. Examples ofpharmaceutically acceptable co-solvents include, but are not limited to,propylene glycol, dipropylene glycol, ethylene glycol, glycerol,ethanol, polyethylene glycols (for example PEG300 or PEG400), andmethanol. In one embodiment, the co-solvent is propylene glycol.

Co-solvent(s), if present, may be included in an amount of from 0.05 to30% (w/w), such as from 1 to 25% (w/w), for example from 1 to 10% (w/w)based on the total weight of the composition.

Compositions for inhaled administration include aqueous, organic oraqueous/organic mixtures, dry powder or crystalline compositionsadministered to the respiratory tract by pressurised pump or inhaler,for example, reservoir dry powder inhalers, unit-dose dry powderinhalers, pre-metered multi-dose dry powder inhalers, nasal inhalers orpressurised aerosol inhalers, nebulisers or insufflators. Suitablecompositions contain water as the diluent or carrier for this purposeand may be provided with conventional excipients such as bufferingagents, tonicity modifying agents and the like. Aqueous compositions mayalso be administered to the nose and other regions of the respiratorytract by nebulisation. Such compositions may be aqueous solutions orsuspensions or aerosols delivered from pressurised packs, such as ametered dose inhaler, with the use of a suitable liquefied propellant.

Compositions for administration topically to the nose (for example, forthe treatment of rhinitis) or to the lung, include pressurised aerosolcompositions and aqueous compositions delivered to the nasal cavities bypressurised pump. Compositions which are non-pressurised and aresuitable for administration topically to the nasal cavity are ofparticular interest. Suitable compositions contain water as the diluentor carrier for this purpose. Aqueous compositions for administration tothe lung or nose may be provided with conventional excipients such asbuffering agents, tonicity-modifying agents and the like. Aqueouscompositions may also be administered to the nose by nebulisation.

A fluid dispenser may typically be used to deliver a fluid compositionto the nasal cavities. The fluid composition may be aqueous ornon-aqueous, but typically aqueous. Such a fluid dispenser may have adispensing nozzle or dispensing orifice through which a metered dose ofthe fluid composition is dispensed upon the application of auser-applied force to a pump mechanism of the fluid dispenser. Suchfluid dispensers are generally provided with a reservoir of multiplemetered doses of the fluid composition, the doses being dispensable uponsequential pump actuations. The dispensing nozzle or orifice may beconfigured for insertion into the nostrils of the user for spraydispensing of the fluid composition into the nasal cavity. A fluiddispenser of the aforementioned type is described and illustrated inInternational Patent Application publication number WO 2005/044354(Glaxo Group Limited). The dispenser has a housing which houses afluid-discharge device having a compression pump mounted on a containerfor containing a fluid composition. The housing has at least onefinger-operable side lever which is movable inwardly with respect to thehousing to move the container upwardly in the housing by means of a camto cause the pump to compress and pump a metered dose of the compositionout of a pump stem through a nasal nozzle of the housing. In oneembodiment, the fluid dispenser is of the general type illustrated inFIGS. 30-40 of WO 2005/044354.

Aqueous compositions containing6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may also be delivered by a pump asdisclosed in International Patent Application publication numberWO2007/138084 (Glaxo Group Limited), for example as disclosed withreference to FIGS. 22-46 thereof, or as disclosed in United Kingdompatent application number GB0723418.0 (Glaxo Group Limited), for exampleas disclosed with reference to FIGS. 7-32 thereof. The pump may beactuated by an actuator as disclosed in FIGS. 1-6 of GB0723418.0.

Dry powder compositions for topical delivery to the lung by inhalationmay, for example, be presented in capsules and cartridges of for examplegelatine, or blisters of for example laminated aluminium foil, for usein an inhaler or insufflator. Powder blend compositions generallycontain a powder mix for inhalation of a maleate salt of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneand a suitable powder base (carrier/d iluent/excipient substance) suchas mono-, di-, or polysaccharides (for example lactose or starch). Drypowder compositions may also include, in addition to the drug andcarrier, a further excipient (for example a ternary agent such as asugar ester for example cellobiose octaacetate, calcium stearate, ormagnesium stearate.

In one embodiment, a composition suitable for inhaled administration maybe incorporated into a plurality of sealed dose containers provided onmedicament pack(s) mounted inside a suitable inhalation device. Thecontainers may be rupturable, peelable, or otherwise openableone-at-a-time and the doses of the dry powder composition administeredby inhalation on a mouthpiece of the inhalation device, as known in theart. The medicament pack may take a number of different forms, forinstance a disk-shape or an elongate strip. Representative inhalationdevices are the DISKHALER™ and DISKUS™ devices, marketed byGlaxoSmithKline.

A dry powder inhalable composition may also be provided as a bulkreservoir in an inhalation device, the device then being provided with ametering mechanism for metering a dose of the composition from thereservoir to an inhalation channel where the metered dose is able to beinhaled by a patient inhaling at a mouthpiece of the device. Exemplarymarketed devices of this type are TURBUHALER™ (AstraZeneca), TWISTHALER™(Schering) and CLICKHALER™ (Innovata.)

A further delivery method for a dry powder inhalable composition is formetered doses of the composition to be provided in capsules (one doseper capsule) which are then loaded into an inhalation device, typicallyby the patient on demand. The device has means to rupture, pierce orotherwise open the capsule so that the dose is able to be entrained intothe patient's lung when they inhale at the device mouthpiece. Asmarketed examples of such devices there may be mentioned ROTAHALER™(GlaxoSmithKline) and HANDIHALER™ (Boehringer Ingelheim.)

Pressurised aerosol compositions suitable for inhalation can be either asuspension or a solution and may contain a maleate salt of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneand a suitable propellant such as a fluorocarbon or hydrogen-containingchlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes,especially 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoro-n-propane or a mixture thereof. The aerosolcomposition may optionally contain additional composition excipientswell known in the art such as surfactants e.g. oleic acid, lecithin oran oligolactic acid or derivative thereof e.g. as described in WO94/21229 and WO 98/34596 (Minnesota Mining and Manufacturing Company)and co-solvents e.g. ethanol. Pressurised compositions will generally beretained in a canister (e.g. an aluminium canister) closed with a valve(e.g. a metering valve) and fitted into an actuator provided with amouthpiece.

Ointments, creams and gels, may, for example, be formulated with anaqueous or oily base with the addition of suitable thickening and/orgelling agent and/or solvents. Such bases may thus, for example, includewater and/or an oil such as liquid paraffin or a vegetable oil such asarachis oil or castor oil, or a solvent such as polyethylene glycol.Thickening agents and gelling agents which may be used according to thenature of the base include soft paraffin, aluminium stearate,cetostearyl alcohol, polyethylene glycols, wool-fat, beeswax,carboxypolymethylene and cellulose derivatives, and/or glycerylmonostearate and/or non-ionic emulsifying agents.

Lotions may be formulated with an aqueous or oily base and will ingeneral also contain one or more emulsifying agents, stabilising agents,dispersing agents, suspending agents or thickening agents.

Powders for external application may be formed with the aid of anysuitable powder base, for example, talc, lactose or starch. Drops may beformulated with an aqueous or non-aqueous base also comprising one ormore dispersing agents, solubilising agents, suspending agents orpreservatives.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may, for example, be formulated fortransdermal delivery by composition into patches or other devices (e.g.pressurised gas devices) which deliver the active component into theskin.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in the conventional manner.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may also be formulated as suppositories,e.g. containing conventional suppository bases such as cocoa butter orother glycerides.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may also be formulated for parenteraladministration by bolus injection or continuous infusion and may bepresented in unit dose form, for instance as ampoules, vials, smallvolume infusions or pre-filled syringes, or in multidose containers withan added preservative. The compositions may take such forms assolutions, suspensions, or emulsions in aqueous or non-aqueous vehicles,and may contain formulatory agents such as anti-oxidants, buffers,antimicrobial agents and/or tonicity adjusting agents. Alternatively,the active ingredient may be in powder form for constitution with asuitable vehicle, e.g. sterile, pyrogen-free water, before use. The drysolid presentation may be prepared by filling a sterile powderaseptically into individual sterile containers or by filling a sterilesolution aseptically into each container and freeze-drying.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may also be formulated with vaccines asadjuvants to modulate their activity. Such compositions may containantibody(ies) or antibody fragment(s) or an antigenic componentincluding but not limited to protein, DNA, live or dead bacteria and/orviruses or virus-like particles, together with one or more componentswith adjuvant activity including but not limited to aluminium salts, oiland water emulsions, heat shock proteins, lipid A preparations andderivatives, glycolipids, other TLR agonists such as CpG DNA or similaragents, cytokines such as GM-CSF or IL-12 or similar agents.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may be employed alone or in combinationwith other therapeutic agents.6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt and the other pharmaceutically activeagent(s) may be administered together or separately and, whenadministered separately, administration may occur simultaneously orsequentially, in any order. The amounts of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt and the other pharmaceutically activeagent(s) and the relative timings of administration will be selected inorder to achieve the desired combined therapeutic effect. Theadministration of a combination of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt with other treatment agents may be byadministration concomitantly in a unitary pharmaceutical compositionincluding both compounds, or in separate pharmaceutical compositionseach including one of the compounds. Alternatively, the combination maybe administered separately in a sequential manner wherein one treatmentagent is administered first and the other second or vice versa. Suchsequential administration may be close in time or remote in time.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may be used in combination with one ormore agents useful in the prevention or treatment of viral infections.Examples of such agents include, without limitation; polymeraseinhibitors such as those disclosed in WO 2004/037818-A1, as well asthose disclosed in WO 2004/037818 and WO 2006/045613; JTK-003, JTK-019,NM-283, HCV-796, R-803, R1728, R1626, as well as those disclosed in WO2006/018725, WO 2004/074270, WO 2003/095441, US2005/0176701, WO2006/020082, WO 2005/080388, WO 2004/064925, WO 2004/065367, WO2003/007945, WO 02/04425, WO 2005/014543, WO 2003/000254, EP 1065213, WO01/47883, WO 2002/057287, WO 2002/057245 and similar agents; replicationinhibitors such as acyclovir, famciclovir, ganciclovir, cidofovir,lamivudine and similar agents; protease inhibitors such as the HIVprotease inhibitors saquinavir, ritonavir, indinavir, nelfinavir,amprenavir, fosamprenavir, brecanavir, atazanavir, tipranavir,palinavir, lasinavir, and the HCV protease inhibitors BILN2061, VX-950,SCH503034; and similar agents; nucleoside and nucleotide reversetranscriptase inhibitors such as zidovudine, didanosine, zalcitabine,abacavir, stavidine, adefovir, adefovir dipivoxil, fozivudine, todoxil,emtricitabine, alovudine, amdoxovir, elvucitabine, and similar agents;non-nucleoside reverse transcriptase inhibitors (including an agenthaving anti-oxidation activity such as immunocal, oltipraz etc.) such asnevirapine, delavirdine, efavirenz, loviride, immunocal, oltipraz,capravirine, TMC-278, TMC-125, etravirine, and similar agents; entryinhibitors such as enfuvirtide (T-20), T-1249, PRO-542, PRO-140,TNX-355, BMS-806, 5-Helix and similar agents; integrase inhibitors suchas L-870,180 and similar agents; budding inhibitors such as PA-344 andPA-457, and similar agents; chemokine receptor inhibitors such asvicriviroc (Sch-C), Sch-D, TAK779, maraviroc (UK-427,857), TAK449, aswell as those disclosed in WO 02/74769, WO 2004/054974, WO 2004/055012,WO 2004/055010, WO 2004/055016, WO 2004/055011, and WO 2004/054581, andsimilar agents; neuraminidase inhibitors such as CS-8958, zanamivir,oseltamivir, peramivir and similar agents; ion channel blockers such asamantadine or rimantadine and similar agents; and interfering RNA andantisense oligonucleotides and such as ISIS-14803 and similar agents;antiviral agents of undetermined mechanism of action, for example thosedisclosed in WO 2005/105761, WO 2003/085375, WO 2006/122011, ribavirin,and similar agents.6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may also be used in combination with oneor more other agents which may be useful in the prevention or treatmentof viral infections for example immune therapies (e.g. interferon orother cytokines/chemokines, cytokine/chemokine receptor modulators,cytokine agonists or antagonists and similar agents); and therapeuticvaccines, antifibrotic agents, anti-inflammatory agents such ascorticosteroids or NSAIDs (non-steroidal anti-inflammatory agents) andsimilar agents.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may be used in combination with one ormore other agents which may be useful in the prevention or treatment ofallergic disease, inflammatory disease, autoimmune disease, for example;antigen immunotherapy, anti-histamines, steroids, NSAIDs,bronchodilators (e.g. beta 2 agonists, adrenergic agonists,anticholinergic agents, theophylline), methotrexate, leukotrienemodulators and similar agents; monoclonal antibody therapy such asanti-IgE, anti-TNF, anti-IL-5, anti-IL-6, anti-IL-12, anti-IL-1 andsimilar agents; receptor therapies e.g. entanercept and similar agents;antigen non-specific immunotherapies (e.g. interferon or othercytokines/chemokines, cytokine/chemokine receptor modulators, cytokineagonists or antagonists, TLR agonists and similar agents).

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may be used in combination with one ormore other agents which may be useful in the prevention or treatment ofcancer, for example chemotherapeutics such as alkylating agents,topoisomerase inhibitors, antimetabolites, antimitotic agents, kinaseinhibitors and similar agents; monoclonal antibody therapy such astrastuzumab, gemtuzumab and other similar agents; and hormone therapysuch as tamoxifen, goserelin and similar agents.

The pharmaceutical compositions according to the invention may also beused alone or in combination with at least one other therapeutic agentin other therapeutic areas, for example gastrointestinal disease. Thecompositions according to the invention may also be used in combinationwith gene replacement therapy.

The invention includes in a further aspect a combination comprising6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, together with at least one othertherapeutically active agent.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical composition and thus pharmaceuticalcompositions comprising a combination as defined above together with atleast one pharmaceutically acceptable diluent or carrier thereofrepresent a further aspect of the invention.

A therapeutically effective amount of a maleate salt of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onewill depend upon a number of factors. For example, the species, age, andweight of the recipient, the precise condition requiring treatment andits severity, the nature of the composition, and the route ofadministration are all factors to be considered. The therapeuticallyeffective amount ultimately should be at the discretion of the attendantphysician. Regardless, an effective amount of a maleate salt of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onefor the treatment of humans suffering from frailty, generally, should bein the range of 0.0001 to 100 mg/kg body weight of recipient per day.More usually the effective amount should be in the range of 0.001 to 10mg/kg body weight per day. Thus, for a 70 kg adult one example of anactual amount per day would usually be from 7 to 700 mg. For intranasaland inhaled routes of administration, typical doses for a 70 kg adultshould be in the range of 1 microgramme to 1mg per day. This amount maybe given in a single dose per day or in a number (such as two, three,four, five, or more) of sub-doses per day such that the total daily doseis the same. Similar dosages should be appropriate for treatment of theother conditions referred to herein.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may also be administered at anyappropriate frequency e.g. 1-7 times per week. The precise dosingregimen will of course depend on factors such as the therapeuticindication, the age and condition of the patient, and the particularroute of administration chosen.

Pharmaceutical compositions may be presented in unit-dose formscontaining a predetermined amount of active ingredient per unit dose.Such a unit may contain, as a non-limiting example, 0.5 mg to 1 g of amaleate salt of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,depending on the condition being treated, the route of administration,and the age, weight, and condition of the patient. Preferred unit-dosagecompositions are those containing a daily dose or sub-dose, as hereinabove recited, or an appropriate fraction thereof, of an activeingredient. Such pharmaceutical compositions may be prepared by any ofthe methods well-known in the pharmacy art.

There is thus further provided a pharmaceutical composition comprising6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, and one or more pharmaceuticallyacceptable diluents or carriers. Optionally, the pharmaceuticalcomposition may further comprise at least one other therapeuticallyactive agent.

There is also provided a process for preparing such a pharmaceuticalcomposition which comprises admixing6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt, with one or more pharmaceuticallyacceptable diluents or carriers.

There is also provided a process for preparing a maleate salt of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onecomprising reacting6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onewith a source of maleate anion (for example, maleic acid e.g. in asuitable solvent) to produce6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt. In one aspect, the process produces a 1:1ratio of maleateanion:6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,in the form of a maleate salt may be prepared by the methodologydescribed hereinafter.

ABBREVIATIONS

The following list provides definitions of certain abbreviations as usedherein. It will be appreciated that the list is not exhaustive, but themeaning of those abbreviations not herein below defined will be readilyapparent to those skilled in the art.

DMF N,N′-Dimethylformamide

DMSO Dimethylsulfoxide

HPLC High performance liquid chromatography

MDAP HPLC Reverse phase HPLC on a C₁₈ column using a two-solventgradient and analysis of the fractions by electrospray massspectroscopy.

SPE Solid phase extraction

min minutes

Stripped Removal of solvent under reduced pressure

TFA Trifluoroacetic acid

rt room temperature

vol volumes

BSA N,O-bis(Trimethylsilyl)acetamide

CPME Cyclopentyl methyl ether

TBME tert-Butyl methyl ether

MeTHF 2-Methyl tetrahydrofuran

NMP N-Methyl pyrrolidinone

DCM Dichloromethane

The synthetic process to make6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,and maleate salts thereof are summarised in Scheme 1.

in which

n=2, m=5 and X═Cl.

Typical reaction conditions for each of the synthetic steps of Scheme 1are provided below:

-   A: Dihydropyran/paratoluene sulfonic acid, e.g. 50° C. for 3-6    hours.-   A1: Dihydropyran/paratoluene sulfonic acid, e.g. 50° C. for 1 hour,    then ammonia/isopropanol, e.g. 60° C. for 4 hours, then add water    and cool to ambient temperature over 12-18 hours.-   A2: BSA in acetonitrile, reflux, cool to 0° C., then tetrahydropyran    acetate in acetonitrile, warm to 10° C., then aqueous sodium    hydrogen carbonate.-   B: Ammonia/isopropanol, e.g. 50° C. for 5 hours, then ambient    temperature for 12-18 hours, then 50° C. for 9 hours.-   C: For Z═O, R^(A)═C₁₋₆alkyl: R^(A)ONa/butanol/dimethoxyethane e.g.    93-110° C. for 12-18 hours.-   C1: N-Bromosuccinimide in dichloromethane e.g. 0-5° C. for 30    minutes then ambient temperature for 0.5-1 hour, then e.g. sodium    methoxide/methanol under nitrogen/60-70° C./12-18 hours, then    TFA/methanol e.g. ambient temperature for 18-65 hours.-   D: N-Bromosuccinimide in dichloromethane e.g. 0-5° C. for 30 minutes    then ambient temperature for 36-48 hours, or N-bromosuccinimide in    chloroform at <5° C. for 4-6 hours.-   E: Sodium methoxide/methanol e.g. reflux 4-6 hours.-   F: TFA/methanol e.g. ambient temperature for 18-65 hours, or    TFA/methanol e.g. ambient temperature for 70-74 hours.-   G: Potassium carbonate/DMF then 50° C. for 1-1.5 hours, then add    (VI), stir 40 minutes, then add (IV)/triethylamine, then ambient    temperature for 18 hours.-   G1: Potassium carbonate/DMF, then 50° C. under nitrogen for 30    minutes, then ambient temperature, add (VI), stir for 20 hours.-   G2: Solution in DMF with N,N′-diisopropylethylamine, then 50° C. for    48 hours, then more (IV) added then further 50° C. for 48 hours.-   H: Hydrogen chloride/methanol, then ambient temperature for 18    hours.

Compounds of formulae (IV), (VI), (XIA), (XII), (XIII), (XIV), and (XV),are either known in the literature or are commercially available, forexample from Sigma-Aldrich, UK, or may be prepared by analogy with knownprocedures, for example those disclosed in standard reference texts ofsynthetic methodology such as J. March, Advanced Organic Chemistry, 6thEdition (2007), WileyBlackwell, or Comprehensive Organic Synthesis(Trost B. M. and Fleming I., (Eds.), Pergamon Press, 1991), eachincorporated herein by reference as it relates to such procedures.

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onein the form of a maleate salt may also be prepared more generally byreacting6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onewith a source of the maleate anion in a suitable solvent e.g isopropylalcohol. A suitable source of the maleate anion is maleic acid or maleicacid salts.

Examples of other protecting groups that may be employed in thesynthetic routes described herein and the means for their removal can befound in T. W. Greene ‘Protective Groups in Organic Synthesis’, 4thEdition, J. Wiley and Sons, 2006, incorporated herein by reference as itrelates to such procedures.

For any of the hereinbefore described reactions or processes,conventional methods of heating and cooling may be employed, for exampletemperature-regulated oil-baths or temperature-regulated hot-blocks, andice/salt baths or dry ice/acetone baths respectively. Conventionalmethods of isolation, for example extraction from or into aqueous ornon-aqueous solvents may be used. Conventional methods of drying organicsolvents, solutions, or extracts, such as shaking with anhydrousmagnesium sulfate, or anhydrous sodium sulfate, or passing through ahydrophobic frit, may be employed. Conventional methods of purification,for example crystallisation and chromatography, for example silicachromatography or reverse-phase chromatography, may be used as required.Crystallisation may be performed using conventional solvents such asethyl acetate, methanol, ethanol, or butanol, or aqueous mixturesthereof. It will be appreciated that specific reaction timestemperatures may typically be determined by reaction-monitoringtechniques, for example thin-layer chromatography and LC-MS.

Where appropriate, individual isomeric forms6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-onemay be prepared as individual isomers using conventional procedures suchas the fractional crystallisation of diastereoisomeric derivatives orchiral high performance liquid chromatography (chiral HPLC).

The absolute stereochemistry of compounds may be determined usingconventional methods, such as X-ray crystallography.

General Experimental Details

Compounds were named using ACD/Name PRO 6.02 chemical naming softwarefrom Advanced Chemistry Developments Inc., Toronto, Ontario, M5H2L3,Canada.

Experimental details of LCMS systems B-D as referred to herein are asfollows:

System B

Column: 30 mm×4.6 mm ID, 3.5 μm Sunfire C₁₈ column

Flow Rate: 3 mL/min.

Temp: 30° C.

UV detection range: 210 to 350 nm

Mass spectrum: Recorded on a mass spectrometer using alternative-scanpositive and negative mode electrospray ionisation

Solvents: A: 0.1% v/v solution of formic acid in water

-   -   B: 0.1% v/v solution of formic acid in acetonitrile

Gradient:

Time (min.) A % B % 0 97 3 0.1 97 3 4.2 0 100 4.8 0 100 4.9 97 3 5.0 973

System C

Column: 50 mm×2.1 mm ID, 1.7 μm Acquity UPLC BEH C₁₈

Flow Rate: 1 mL/min.

Temp: 40° C.

UV detection range: 210 to 350 nm

Mass spectrum: Recorded on a mass spectrometer using alternative-scanpositive and negative mode electrospray ionisation

Solvents: A: 10 mM ammonium bicarbonate in water adjusted to pH10 withammonia solution

-   -   B: acetonitrile

Gradient:

Time (min.) A % B % 0 99 1 1.5 3 97 1.9 3 97 2.0 0 100

System D

Column: 50 mm×4.6 mm ID, 3.5 μm XBridge C₁₈ column

Flow Rate: 3 mL/min.

Temp: 30° C.

UV detection range: 210 to 350 nm

Mass spectrum: Recorded on a mass spectrometer using alternative-scanpositive and negative mode electrospray ionisation

Solvents: A: 10 mM ammonium bicarbonate in water adjusted to pH10 withammonia solution

-   -   B: acetonitrile

Gradient:

Time (min.) A % B % 0 99 1 0.1 99 1 4.0 3 97 5.0 3 97

Chromatographic purification was typically performed using pre-packedsilica gel cartridges. The Flashmaster II is an automated multi-userflash chromatography system, available from Argonaut Technologies Ltd,which utilises disposable, normal phase, Solid Phase Extraction (SPE)cartridges (2 g to 100 g). It provides quaternary on-line solvent mixingto enable gradient methods to be run. Samples are queued using themulti-functional open access software, which manages solvents,flow-rates, gradient profile and collection conditions. The system isequipped with a Knauer variable wavelength UV-detector and two GilsonFC204 fraction-collectors enabling automated peak cutting, collectionand tracking.

Solvent removal using a stream of nitrogen was performed at 30-40° C. ona GreenHouse Blowdown system available from Radleys DiscoveryTechnologies Saffron Walden, Essex, CB11 3AZ, UK

¹H NMR spectra were recorded in either CDCl₃ or DMSO-d₆ on either aBruker DPX 400 or Bruker Avance DRX or Varian Unity 400 spectrometer allworking at 400 MHz. The internal standard used was eithertetramethylsilane or the residual protonated solvent at 7.25 ppm forCDCl₃ or 2.50 ppm for DMSO-d₆.

Mass directed autopreparative HPLC was undertaken under the conditionsgiven below. The UV detection was an averaged signal from wavelength of210 nm to 350 nm and mass spectra were recorded on a mass spectrometerusing alternate-scan positive and negative mode electrospray ionization.

Method A

Method A was conducted on an XBridge C₁₈ column (typically 150 mm×19 mmi.d. 5 μm packing diameter) at ambient temperature. The solventsemployed were:

A=10 mM aqueous ammonium bicarbonate adjusted to pH 10 with ammoniasolution.

-   -   B=acetonitrile.

Intermediates Intermediate 1:2,6-Dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine

To 2,6-dichloropurine (25.0 g) (commercially available from, forexample, Aldrich) was added ethyl acetate (260 mL), followed byp-toluenesulfonic acid (0.253 g). The mixture was heated to 50° C. andthen 3,4-dihydro-2H-pyran (16.8 g) (commercially available from, forexample, Aldrich) was added. The reaction mixture was then heated at 50°C. for 4 hours. The reaction mixture was evaporated in vacuo to give thetitle compound as a yellow solid (36.9 g).

¹H NMR (CDCl₃): 8.35 (1H, s), 5.77 (1H, dd), 4.20 (1H, m), 3.79 (1H, m),2.20-1.65 (6H, m).

Intermediate 1A

Acetic acid (1.2 L, 1 eq) and pyridinium p-toluene sulfonate (530 g, 0.1eq), were dissolved in dichloromethane (6 L). The solution was cooled to0° C. A solution of the dihydropyran (2.52 L, 1.35 eq) indichloromethane (2.5 L) was charged cautiously over at least 15 mins.keeping the temperature below 5° C. Once the addition was complete, thesolution was warmed to 20° C. and stirred overnight. Water (5.0 L) wascharged and the resultant biphase was stirred vigorously before removingthe aqueous layer. The organic phase was then washed with saturatedsodium bicarbonate solution (5.0 L) and dried over magnesium sulfate.The dried organic phases were concentrated on the rotary evaporator,reducing the pressure to 20 mbar at 50° C. to ensure removal of DCM andexcess dihydropyran. The product was afforded as a colourless toslightly yellow liquid (2.61 kg, 95% theoretical yield).

Intermediate 2: 2-Chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

2,6-Dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (36.9 g) (forexample, as prepared for Intermediate 1) was heated with 2M ammonia inisopropanol (250 mL) at 50° C. for 5 hours. After standing at ambienttemperature overnight, a further quantity of 2M ammonia in isopropanol(100 mL) was added to break up the resultant cake and the reactionmixture was heated for a further 9 hours until the reaction wascomplete. To the reaction mixture was added water (70 mL) and the yellowsolid filtered off. The solid was washed with isopropyl alcohol:water(5:1 (v/v), 60 mL) and then air-dried under suction to give a firstcrop. The filtrate was re-filtered after standing overnight to isolateprecipitate and both solids were dried in vacuo. The first crop was purewith the second crop material showing a very minor impurity (isolatedbroad signal 3.5 ppm not seen in first crop) but was otherwiseidentical. Solid first crop (28.4 g), solid second crop (3.42 g).

¹H NMR (CDCl₃): 8.01 (1H, s), 5.98 (2H, broad s), 5.70 (1H, dd), 4.16(1H, m), 3.78 (1H, m), 2.15-1.60 (6H, overlapping m).

Intermediate 2 (Alternative Method):2-Chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

To a solution of 2,6-dichloropurine (25 g) (commercially available from,for example, Aldrich) in dry ethyl acetate (200 mL) was addedp-toluenesulfonic acid monohydrate (235 mg) (commercially availablefrom, for example, Aldrich). The reaction was heated to 50° C. and3,4-dihydro-2H-pyran (18.1 ml) (commercially available from, forexample, Aldrich) was added in one go. The reaction was allowed to stirat 50° C. for 1 hour and the solvent was removed under reduced pressure.This afforded a yellow solid. A suspension of this solid (˜36 g) in 2.0Mammonia in isopropanol (460 mL) was heated under nitrogen at 60° C. for4 hours with an attached condenser. The reaction was poured into water(50 mL) and left to cool overnight. The precipitate was filtered anddried on a rotary evaporator (60° C.) for 30 minutes to afford the titlecompound as an off-white solid, 31 g (93%, 2 steps).

MS calcd for (C₁₀H₁₂ClN₅O)⁺=254, 256

MS found (electrospray): (M)⁺=254, 256 (3:1)

¹H NMR ((CD₃)₂SO): δ 8.43 (1H, s), 7.82 (2H, s), 5.55 (1H, dd), 4.00(1H, m), 3.69 (1H, m), 2.21 (1H, m), 1.95 (2H, m), 1.74 (1H, m), 1.56(2H, m).

Intermediate 3: 2-Fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

N,O-bis(trimethylsilyl)acetamide (975 mL, 3.988 mol) was added to astirred suspension of 2-fluoro-1H-purin-6-amine (200 g, 1.306 mmol)(commercially available from, for example, AlliedSignal) in anhydrousacetonitrile (4 L) in a 10 L controlled lab reactor and the resultingmixture heated to reflux and maintained at that temperature for 2 hours.The circulator was then re-programmed and the reaction mixture cooled to0° C. A solution of tetrahydropyranyl acetate (preparation described inTetrahedron Letters, 2006, 47(27), 4741 and also described inIntermediate 1A) (282 g, 1.959 mol) in anhydrous acetonitrile (500 mL)was then added slowly via a dropping funnel followed by trimethylsilyltrifluoromethanesulfonate (283 mL, 1.567 mol) dropwise via a droppingfunnel. No significant exotherm was observed. The circulator temperaturewas re-adjusted to 10° C. and stirring maintained for a further 1 hour.The mixture was then quenched by addition of 1M sodium carbonate (4 L).A solid precipitate was observed and the pH checked to be basic.Additional water was added to the suspension (1 L) and on standing thelayers separated with the aqueous layer containing significant solidinorganics. The majority of the aqueous and inorganic solid wasseparated. The organic layer still contained significant solid and wascooled to 0° C. with stirring to encourage further precipitation. Thesolid was the collected by filtration and the pad was washed very wellwith water then dried in vacuo at 40° C. overnight to give the titlecompound as a cream coloured solid (152.8 g).

LCMS (System D): t_(RET)=1.71 min; MH⁺=238

Intermediate 4:2-{[(1S)-1-Methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

Method A

Sodium tert-butoxide (48.5 g, 505 mmol) was added portionwise to(S)-2-pentanol (185 ml) (commercially available from, for example,Julich Chiral Solutions) at room temperature stirred until homogeneous(Note: reaction is exothermic).2-Chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (32 g, 126 mmol)(for example, as prepared for Intermediate 2) was added and the reactionmixture heated at 70° C. for 72 hours. The reaction was cooled to roomtemperature and partitioned between ethyl acetate (500 mL) and water(500 mL). The organic phase was washed with saturated sodium chloridesolution (100 mL), dried (MgSO₄), filtered and evaporated. The residuewas triturated with ether and the solid material filtered. Theprecipitate was re-washed with ether and the filtrates combined andevaporated. The crude material (approximately 30 g) was dissolved inDMSO:methanol (1:1) and purified by chromatography on a reverse phase(C₁₈) column (330 g) using a gradient of 25-65% acetonitrile (+0.1%TFA)-water(+0.1% TFA) over 8 column volumes, the fractions wereimmediately neutralised with saturated aqueous sodium carbonatesolution. Appropriate fractions were combined and partitioned betweendichloromethane and saturated aqueous sodium hydrogen carbonate. Theorganic phase was dried by passage through a hydrophobic frit, filteredand evaporated to give the title compound as a pale cream foam (14.97g).

LCMS (System B): t_(RET)=2.21 min; MH⁺ 306

Method B

Sodium tert-butoxide (206 g, 2.144 mol) was added to (S)-2-pentanol (720mL, 6.58 mol) (commercially available from, for example, Julich ChiralSolutions) in a 2 L round bottomed flask. The mixture was stirred at 50°C. until all the sodium tert-butoxide had dissolved.2-Fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (for example, asprepared for Intermediate 3) (130 g, 548 mmol) was then added inportions over 5 minutes. After 3 hours, LCMS analysis indicated completeconsumption of the starting material and the mixture was poured intoice/water (3 L) and then extracted with methyl tert-butyl ether. Thisresulted in emulsion formation and the mixture was filtered throughCelite and the organic phase was separated. The aqueous layer was thentreated with solid NaCl and then re-extracted with methyl tert-butylether. The organic extracts were combined and washed with brine, driedover magnesium sulfate, filtered and then evaporated to yield the titlecompound as a pale brown gum (158.59 g).

LCMS (System D): t_(RET)=2.65 min; MH⁺ 306

Intermediate 5:8-Bromo-2-{[(1S)-1-methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

N-Bromosuccinimide (12.16 g, 68.3 mmol) was added portionwise over 5minutes to a stirred solution of2-{[(1S)-1-methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(14.9 g, 48.8 mmol) (for example, as prepared for Intermediate 4) inchloroform (80 mL) at <5° C. under an atmosphere of nitrogen. Thereaction mixture was stirred at <5° C. for 5 hours then washed withsaturated sodium hydrogen carbonate solution (80 mL) then water (80 mL).The foam was dissolved in dichloromethane (50 mL) and washed with water(50 mL) then brine (50 mL). The combined aqueous phases were washed withdichloromethane (50 mL). The combined organic layers were dried througha hydrophobic frit, and the solvent removed in vacuo to yield the titlecompound as an orange foam (18.5 g).

LCMS (System D): t_(RET)=3.06 min; MH⁺ 384/386

Intermediate 5 (Alternative Method):8-Bromo-2-{[(1S)-1-methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

2-{[(1S)-1-Methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(1050 g) was dissolved in DCM (10.5 L) to give a yellow/orange solutionwhich was cooled to 0° C. N-Bromosuccinimide (922 g, 1.5 eq) was chargedin 3 equal portions 20 mins. apart and the resultant reaction mixturewas stirred at 0-5° C. for 4 hours. The reaction was then quenched byaddition of a solution of 500 g sodium thiosulfate pentahydrate in 5.0 Lwater. The resultant biphase was mixed thoroughly at 20° C. and then thephases were separated. The organics were washed again with a solution of500 g sodium thiosulfate pentahydrate in 5.0 L water then 500 gdipotassium phosphate in 5.0 L water and finally with 5.0 L water. Theorganic phase was dried over magnesium sulfate (822 g) and evaporated ona rotary evaporator until foaming became prohibitive. The mixture wasthen solvent-exchanged into methanol by repeated addition and removal ofmethanol until sufficient DCM had been removed (as confirmed by NMR).The product was afforded as a red/brown gum containing entrained solvent(1.28 kg corrected for solvent, 96% theoretical yield).

Intermediate 6:2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

8-Bromo-2-{[(1S)-1-methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(for example, as prepared for Intermediate 5) (7.1 g, 18.48 mmol) wasdissolved in anhydrous methanol (70 mL) and a solution of sodiummethoxide (25%) in methanol (8 mL) was added dropwise under anatmosphere of nitrogen. The solution was heated to reflux at 90° C. for4 hours under an atmosphere of nitrogen. Additional sodium methoxide inmethanol (25% solution, 3 mL) was added and the reaction was stirred at60° C. for a further 16 hours. An additional portion of sodium methoxidein methanol (25% solution, 5 mL) was added and the reaction was stirredat 90° C. for a further 7 hours. The solvent was removed on the rotaryevaporator and the crude product was partitioned between ethyl acetate(75 mL) and saturated ammonium chloride solution (75 mL). The organiclayer was washed with brine (75 mL). The solvent was removed on therotary evaporator to yield the title compound as a pale orange foam (6g).

LCMS (System C): t_(RET)=1.14 min; MH⁺ 336, 337

Intermediate 7:2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine,trifluoroacetate Salt

2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(6 g, 17.89 mmol) (for example as prepared for Intermediate 6) wasdissolved in methanol (50 mL). Trifluoroacetic acid (20.67 mL, 268 mmol)was added dropwise, and the mixture stirred at 20° C. for 72 hours underan atmosphere of nitrogen. The solvent was removed in vacuo, and theresulting solid was washed with ethyl acetate and filtered. The filtratewas stripped and the residue washed with ethyl acetate. The combinedsolid residues were dried in the vacuum oven for 2 hours to give thetitle compound as an off white solid (5.3 g).

LCMS (System C): t_(RET)=0.76 min; MH⁺ 252, 253

Intermediate 7 (Alternative Method):2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine,trifluoroacetate Salt

8-Bromo-2-{[(1S)-1-methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(1.26 kg, corrected for residual solvent) was dissolved in anhydrousmethyl tetrahydrofuran (MeTHF) (11.4 L) and 25% sodium methoxide inmethanol (2.65 L, 3.5 eq) was added. The resultant reaction mixture washeated to 65+/−5° C. for 3 hrs. The complete reaction was cooled to roomtemperature and washed with 20% w/v aqueous ammonium chloride solution(2×6.3 L) and brine (6.3 L). The organic phase was dried with MgSO₄ (1.8kg) and filtered, washing through with MeTHF (6.3 L). The combinedorganic phases were evaporated to 6.3 L by vacuum distillation. MeOH(2.5 L) and TFA (1.26 L, 5 eq) were added and the mixture heated to 60°C. for 1.5 hours. Cyclopentyl methyl ether (CPME) (6.3 L) was added andthe mixture reduced to 6.3 L by vacuum distillation. CPME (6.3 L) wasagain added and the reaction further concentrated to 6.3 L, when solidsprecipitated. The slurry was cooled to 10° C. then aged for 30 min. Theproduct was filtered and washed with TBME (2×3.8 L) and dried in vacuoat 40° C. to afford a white solid (886 g, 74% theoretical yield).

Intermediate 8:9-(5-Chloropentyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine

2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine,trifluoroacetate salt (600 mg, 1.642 mmol) (for example, as prepared forIntermediate 7) and potassium carbonate (567 mg, 4.11 mmol) were stirredat 60° C. in DMF (10 mL) for 1 hour under nitrogen. The reaction wascooled to room temperature when 1-bromo-5-chloropentane (commerciallyavailable, for example, from Aldrich) (0.216 mL, 1.642 mmol) andtriethylamine (0.343 mL, 2.464 mmol) were added and the mixture stirredat 20° C. under nitrogen for 16 hours. The mixture was then diluted withwater (10 mL) and brine (10 mL) and extracted with dichloromethane (2×10mL). The combined organic extracts were evaporated and the residuedissolved in dichloromethane and purified by column chromatography usingthe Flashmaster II (70 g aminopropyl cartridge) with a 0-100% ethylacetate in cyclohexane gradient over 40 minutes. The appropriatefractions were combined and evaporated in vacuo to give the titlecompound as a yellow gum (430 mg).

LCMS (System D): t_(RET)=4.15min; MH⁺=356, 358

Intermediate 8 (Alternative Method as Sulfuric Acid Salt):9-(5-Chloropentyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine,sulfuric Acid Salt

Sodium hydroxide (2M, 2.52 L, 2.3 eq.) was added to a solution of2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine,trifluoroacetate salt (800 g, 1.0 eq.) in NMP (3.08 L).1-Bromo-5-chloropentane (432 mL, 1.5 eq.) was added. The reactionmixture was heated to 50° C. for 6 h. The reaction mixture was cooled to20-25° C. Ethyl acetate (8.0 L) was added, followed by water (1.6 L).After stirring for 10 minutes, the phases were separated and the organicphase was then washed with water (1.6 L). The ethyl acetate phase wasfurther diluted with ethyl acetate (4.0 L) and heated to 50° C. Sulfuricacid (117 mL, 1 eq.) was added dropwise. The reaction mixture was cooledto 10° C. over 1.5 hours and aged for half an hour. The product wasisolated by filtration as a white solid, washed on the filter with ethylacetate (2.4 L) and dried under reduced pressure at 40° C. (570 g, 57%theoretical yield).

Intermediate 9:2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-[5-(1-piperidinyl)pentyl]-9H-purin-6-amine

9-(5-Chloropentyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine(for example, as prepared for Intermediate 8) (80 mg, 0.225 mmol),triethylamine (0.031 mL, 0.225 mmol) and piperidine (0.045 mL, 0.45mmol) were suspended in DMF (3 mL) and the mixture heated to 70° C. for18 hours. The solvent was removed and the residue partitioned betweendichloromethane (4 mL) and saturated sodium bicarbonate (4 mL). Theaqueous phase was re-extracted with further dichloromethane and thecombined organic extracts were concentrated and the residue dissolved in1:1 methanol:DMSO (1 mL) and purified by MDAP (Method A). Theproduct-containing fractions were combined and evaporated under a streamof nitrogen to give the title compound (47.2 mg).

LCMS (System D): tRET=3.11 min; MH+=405

EXAMPLES Reference Example 16-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one

A solution of hydrogen chloride in dioxane (4M, 0.71 mL) was added to asolution of2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9-[5-(1-piperidinyl)pentyl]-9H-purin-6-amine(for example, as prepared for Intermediate 9) (0.046 g, 0.126 mmol) inmethanol (3 mL). The resultant mixture was allowed to stand overnight atroom temperature and then blown down under nitrogen. The residue wasdissolved in methanol and loaded onto a 2 g aminopropyl SPE cartridge(pre-conditioned with methanol), eluted with methanol and the resultantsolution blown down under nitrogen to give the title compound as ayellow solid (40.97 mg).

LCMS (System D): tRET=2.70 min; MH+=391

A similarly prepared sample (1.7 g) was recrystallised from ethylacetate (approximately 50 mL). The crystals were collected, washed withice-cold ethyl acetate (15 mL) and dried in vacuo at 50° C. for 3 hoursto give the title compound as a cream crystalline solid (1.33 g).

Melting point onset (DSC): 207.4° C. (see FIG. 2)

XRPD: (see FIG. 1 and Table 1)

Reference Example 1: (Alternative Method)6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one

9-(5-Chloropentyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine,sulfuric acid salt (254 g, 1.0 eq) was dissolved in DMSO (1.27 L) andpiperidine (280 mL, 5 eq). The reaction mixture was heated to 70±3° C.for 15.5 h. The reaction mixture was cooled to 20±3° C. Toluene (2.5 L)was added, followed by water (1.25 L). After stirring for 10 minutes,the phases were separated and the upper toluene phase was washed withwater (0.5 L). A solution of hydrochloric acid (2.24 mol) in water (1.5L) was added. The mixture was stirred for 10 minutes and then allowed toseparate with the lower (aqueous) phase retained. The aqueous solutionwas heated to 50±3° C. for 17 h and then cooled to 20±3° C. Aqueoussodium hydroxide (2M, ca. 840 mL) was added dropwise until the solutionhad a pH of 10-11. The resulting suspension was cooled to 10±3° C., agedfor a further 30 min. then filtered. The cake was washed with water (7.6L) and the product was dried under reduced pressure at 60° C. with anitrogen bleed to constant weight (207 g, 95% th).

Polymorphism

X-ray powder diffraction (XRPD) and differential scanning calorimetry(DSC) were performed on6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneaccording to the following methods.

XRPD

XRPD data were acquired on a PANalytical X'Pert Pro powderdiffractometer, equipped with an X'Celerator detector. The acquisitionconditions were: radiation: Cu Kα, generator tension: 40 kV, generatorcurrent: 45 mA, start angle: 2.0° 2θ, end angle: 40.0° 2θ, step size:0.016° 2θ. The time per step was 31.750 s. The sample was prepared bymounting a few milligrams of sample on a Si wafer (zero background)plate, resulting in a thin layer of powder.

Characteristic peak positions and calculated d-spacings are summarisedin Table 1. These were calculated from the raw data using Highscoresoftware. The experimental error in the peak positions is approximately±0.1° 2θ. Relative peak intensities will vary due to preferredorientation.

TABLE 1 Characteristic XRPD Peak Positions for Solid-state Form 1 of6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one Form 1 2θ/°d-spacing/Å 5.0 17.6 10.0 8.8 12.7 7.0 13.5 6.5 13.8 6.4 16.6 5.3 18.94.7 20.0 4.4 22.2 4.0 23.3 3.8 24.2 3.7 26.1 3.4

A representative XRPD diffractogram of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneis shown in FIG. 1.

DSC

The DSC thermogram was obtained using a TA Instruments calorimeter. Thesample was weighed into an aluminium pan, a pan lid placed on top andlightly crimped without sealing the pan. The experiment was conductedusing a heating rate of 10° C. min⁻¹.

A representative DSC thermogram of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneis shown in FIG. 2.

Example 26-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,Maleate Salt

Preparation 1

6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one(for example, as prepared for Reference Example 1) (0.384 g, 0.98 mmol)was dissolved in isopropyl alcohol (4.6 mL, 12 vols) and heated to 40°C. Maleic acid (0.114 g, 0.98 mmol) was added. A clear solution wasobtained. During cooling to room temperature, precipitation occurred.The slurry was filtered, washing with isopropyl alcohol (5 mL) and driedunder reduced pressure at 40° C. to constant weight.6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,maleate salt (0.305 g, 61% th) was obtained as a white solid.

¹H NMR confirms a 1:1 ratio of maleic acid:6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one.¹H NMR (400 MHz, DMSO-d₆) δ ppm, 9.85 (1H, s, (CH₂)₃NHCO), 8.85 (1H, brs, NH⁺), 6.39 (2H, s, NH₂), 6.02 (2H, s, HO₂C(CH)₂), 5.00 (1H, m, J=6.2Hz, CH₃CH), 3.68 (2H, t, J=6.8, Hz NCH₂), 3.40 (2H, m, NCH₂), 2.98 (2H,m, J=8.1 Hz NCH₂), 2.82 (2H, br s, NCH₂), 1.85-1.24 (16H, m, 8×CH₂),1.21 (3H, d, J=6.1 Hz, CHCH₃), 0.89 (3H, t, J=7.3 Hz, CH₂CH₃), 2.5(solvent (DMSO)).

Preparation 2

A solution of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one(for example, as prepared for Reference Example 1) (1.46 g, 3.74 mmol)in isopropyl alcohol (14.6 mL, 10 vols) was clarified (filtered at roomtemperature through a BondElut cartridge) and then heated toapproximately 50° C. A solution of maleic acid (0.434 g, 3.74 mmol) inisopropyl alcohol (2.9 mL, 2 vols) was added. The resulting solution wasthen seeded and cooled to 45° C. Further seed was added. The resultingslurry was cooled to room temperature and held overnight (approximately16 hours), then cooled in an ice/water bath for 30 minutes. The slurrywas filtered, washing with isopropyl alcohol (4.5 mL, 3 vols and then 3mL, 2 vols). The product was dried under reduced pressure at 40° C. toconstant weight to give6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,maleate salt (1.305 g, 69% th).

Analysis by XRPD (FIG. 3) indicated this sample to be crystalline.

Biological Data

The compound of Reference Example 1 was tested for in vitro biologicalactivity in accordance with the following assays, or similar assays:

Assay for the Induction of Interferon-α Using Cryopreserved HumanPeripheral Blood Mononuclear Cells (PBMCs)

Compound Preparation

The compound of Reference Example 1 was dissolved in DMSO. Serial 2-folddilutions with DMSO were prepared and 0.25 μl dispensed into 384-wellclear Greiner polypropylene plates.

Preparation of PBMCs

Blood samples of up to 200 mL were obtained from healthy human donors.Whole blood in 25 mL volumes was overlaid onto 15 mL Ficoll gradients inLeucosep tubes, and centrifuged at 1000 g for 20 min. Cells in the bandat the plasma/histopaque interface were carefully removed and washedtwice with PBS (centrifuged at 400 g for 5 min to harvest). The finalpellet was resuspended in freezing medium (90% Heat-inactivated serum,10% DMSO) to a cell concentration of 4×10⁷ cells/mL. The resuspendedcells were then cryopreserved (frozen) using a rate controlled freezer,and stored at −140° C. for up to 4 months.

Incubation and Assay for Interferon-α

Immediately prior to assay, vials of cryopreserved (frozen) PBMCs werethawed rapidly in a water bath at 37° C. A 1:10 dilution of the cells intrypan blue was prepared and counted. The PBMCs were then diluted ingrowth media [RPMI 1640 containing 10% fetal calf serum (Invitrogen),Penicillin+Streptavidin (Gibco cat. #25030-024, 1:50), L-Glutamine 2 mM,and 1000 units/mL recombinant human IFN-gamma (Preprotech catalogue#300-02)] to a density of 1×10⁶ cells/mL, and 50 μL/well dispensed to384-well clear Greiner polypropylene plates containing 0.25 μL DMSO ortest compound in 0.25 μL DMSO. Top final concentration of compound wastypically 50 μM or 5 μM (to obtain curve fit for highly activecompounds). Plates were incubated for 24 hours at 37° C. in 5% CO₂.

A multi-isoform immunoassay was used to quantify IFN-α in PBMCsupernatants. Rabbit polyclonal antibody against human IFN-α (cataloguenumber 31101, Stratech Scientific) was diluted 1:10000 in assay buffer(RPMI 1640 containing 10% fetal calf serum, Invitrogen) and 20 μL wasadded to each well of an MSD (Meso-Scale Discovery) single small-spot384-well GAR (goat anti-rabbit antibody coated) plate. The plate wasincubated for 1 hour at room temperature with vigorous shaking.Following three washes with PBS, 20 μL of cell supernatant were added toeach well of the plate. The plate was then incubated for 1 hour at roomtemperature with vigorous shaking. A pair of monoclonal antibodies toIFN-α (catalogue numbers 21100 and 21112, Stratech Scientific) werelabelled with sulfo-TAG (MSD), diluted 1:1000 in assay buffer and 20 μLadded to each well of the plate. The plate was further incubated for 1hour at room temperature with vigorous shaking. Following three washeswith PBS, 30 μl of ×2 T buffer (MSD) was added to each well and theplate was read on an MSD Sector 6000 plate reader.

Data were normalised to internal plate controls of 1 μM resiquimod(n=16) and DMSO (n=16). pEC₅₀ values were derived by 4-parameter curvefit with IRLS in ActivityBase, from 11-point, two-fold serial dilutionof test compounds.

Results

Reference Example 1 had a mean pEC₅₀ of >8.3

Assay for the Induction of Interferon-α and TNF-α Using Fresh HumanPeripheral Blood Mononuclear Cells (PBMCs)

Compound Preparation

The compound of Reference Example 1 was dissolved and serially dilutedin DMSO to give 100× the required concentration range using a Biomek2000. 1 μL of test compound was transferred into 96-well tissue cultureplates using a Biomek FX. Each compound was assayed in duplicate foreach donor. Each plate contained a dilution series of the TLR7/8 agonistresiquimod as standard and Column 11 contained 1 μL of 200 μM resiquimod(giving a 2 μM final concentration, used to define the approximatemaximal response to resiquimod).

Preparation of PBMCs

Blood samples from two human donors were collected into sodium heparin(10 U/mL). 25 mL volumes of whole blood were overlaid onto 15 mLHistopaque in Leucosep tubes which were centrifuged at 800 g for 20 minand the band at the plasma/histopaque interface carefully removed. Thecollected cells were centrifuged at 2500 rpm for 10 min and the pelletresuspended in 10 mL of media (RPMI 1640 (Low endotoxin) supplementedwith 10% v/v foetal calf serum (FCS, low endotoxin) 100 U/mL penicillinG, 100 μg/mL streptomycin, 10 mM L-glutamine and 1× non-essential aminoacids). A 1:20 dilution of the cells was prepared using trypan blue andthe cells counted using a haemocytometer. The PBMCs were diluted to givea final concentration of 2×10⁶ per mL and 100 μL of this cellssuspension was added to wells containing 1 μL of diluted test compound.

Incubation and Assays for Interferon-α and TNF-α

The cell preparations were incubated for 24 hr (37° C., 95% air, 5% CO₂)after which a sample of the supernatant was removed using the Biomek FXand assayed for both IFN-α and TNF-α using the MSD (Mesoscale Discovery)electrochemiluminescence assay platform. The IFN-α assay was carried outsimilarly to that described above. The TNF-α assay was carried out asper kit instructions (Cat No K111BHB).

Cytokine released was expressed as a percentage of the 2 μM resiquimodcontrol (column 11). This percentage was plotted against compoundconcentration and the pEC₅₀ for the response determined by non-linearleast squares curve fitting. For the IFN-α responses generally a 4parameter logistic model was selected. For the TNF responses where aclear maximum response was obtained (i.e. a well defined plateau in theresponse was observed) then a 4 parameter model was generally used. Ifthe upper asymptote of the curve wasn't well defined then the curvefitting was generally constrained to a maximal response of 100% (i.e. tothe response to 2 μM resiquimod) or to the response of the highestconcentration tested if this was greater than the resiquimod response.Some curves were bell shaped for one or both cytokines and the cytokinedata on the down slope of the bell shaped response (i.e. concentrationsabove those giving the maximal response) were generally excluded fromthe fit, usually with the exception of the concentration immediatelyabove the peak response. Curve fitting thus concentrated on the up slopeof the dose response curve.

Results

Reference Example 1 showed a mean pEC₅₀ for induction of IFN-α and TNF-αof ≧9 and ≦6.5 respectively.

Allergen-Driven Cytokine Assay Using Fresh Human Peripheral BloodMononuclear Cells (PBMCs) from Atopic Volunteers

An assay based on co-culture of atopic human donor derived peripheralblood mononuclear cells (PBMCs) with allergen and test compounds wasdeveloped. After 5-6 days culture, cell supernatants were assayed for arange of cytokines.

Compound Preparation

The compound of Reference Example 1 was dissolved in DMSO, then seriallydiluted in growth medium (RPMI 1640 medium supplemented with 100 U/mLpenicillin G, 100 μg/mL streptomycin, 10 mM L-glutamine) to give 4× therequired concentration range in the presence of 0.04% DMSO. Eachcompound was assayed in triplicate at all concentrations.

Preparation of PBMCs

Defibrinated human blood from volunteers known to be allergic to Timothygrass was centrifuged at 2500 rpm for 15 minutes. The upper layer ofserum was collected and heat-inactivated at 56° C. for 30 minutes(HI-autologous serum). The lower layer of cells was resuspended in 50 mLPBS (+Ca +Mg), 25 mL diluted blood were overlaid onto 20 mL Lymphoprepin 50 ml tubes then centrifuged at 2500 rpm for 20 minutes at RT. Theband at the serum/Lymphoprep interface was carefully removed. Thecollected cells were washed with PBS and re-suspended at 4×10⁶ per mL ingrowth medium with HI-autologous serum. PBMCs were seeded at 0.4×10⁶cells per well in flat-bottomed 96 well plates in the presence of 10ug/mL Timothy Grass antigen (Alk Abello) and test compounds atappropriate concentrations in a total volume of 200 μL.

Incubation and Cytokine Assays

Plates were incubated at 37° C. in 5% CO₂ for up to 6 days. The cellmedium from each well was harvested and stored at −20° C. prior toanalysis. Cytokines and chemokines in supernatants were detected usingMeso Scale Discovery 10 spot plates for Human TH1/Th2 cytokines.

In the above assay, data from separate studies with PBMCs from threeallergic donors showed Reference Example 1 to reduce production of theTh2 cytokines IL-5 and IL-13 in a dose response manner with ≧50%reduction observed at 0.04 μM compared to the allergen control.

Reference Example 1 was also tested for in vivo biological activity inthe following model:

Assay for the Induction of Interferon-α Following Intranasal Dosing inthe Mouse

The compound of Reference Example 1 was dissolved in 0.2% Tween 80 insaline and administered intranasally (5 μL in total between thenostrils) to female BALB/c mice (n=6) under general anaesthesia. Animalswere euthanased 2 hours after dosing and a terminal blood sample wastaken and serum levels of Interferon-a measured using an ELISA assay.

In this model Reference Example 1 showed mean serum levels ofInterferon-α of 21029 pg/mL. No Interferon-a was detected in vehicletreated controls.

Stability Testing

6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,maleate salt exhibited no significant degradation under conditionsspecified in Quality Guidelines Q1A(R2) (Stability Testing of New DrugSubstances and Products) and Q1B (Photostability Testing of New DrugSubstances and Products) laid down by the International Conference forHarmonisation of Technical Requirements for Registration ofPharmaceuticals for Human Use (ICH).

1. A compound which is6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one:

in the form of a maleate salt. 2-5. (canceled)
 6. A vaccine adjuvantcomprising a compound according to claim
 1. 7. An immugenic compositioncomprising an antigen or antigen composition and a compound according toclaim
 1. 8. A vaccine composition comprising an antigen or antigencomposition and a compound according to claim
 1. 9-13. (canceled)
 14. Amethod of treatment of allergic diseases and other inflammatoryconditions, infectious diseases, and cancer, which method comprisesadministering to a human subject in need thereof, a therapeuticallyeffective amount of a compound according to claim
 1. 15. A method oftreatment of allergic rhinitis, which method comprises administering toa human subject in need thereof, a therapeutically effective amount of acompound according to claim
 1. 16. A method of treatment of asthma,which method comprises administering to a human subject in need thereof,a therapeutically effective amount of a compound according to claim 1.17. A pharmaceutical composition comprising a compound according toclaim 1, and one or more pharmaceutically acceptable diluents orcarriers.
 18. A method of treating or preventing disease comprising theadministration to a human subject suffering from or susceptible todisease, a vaccine composition comprising an antigen or antigencomposition and a compound according to claim
 1. 19. A method oftreating or preventing disease comprising the administration to a humansubject suffering from or susceptible to disease, an immugeniccomposition comprising an antigen or antigen composition and a compoundaccording to claim
 1. 20. A pharmaceutical composition according toclaim 17 which is administered by intranasal or inhaled administration.